Maize (Zea mays) is an important crop in many areas of the world. The methods of biotechnology have been applied to this crop in order to produce maize with desirable traits. One such desirable trait is herbicide tolerance. Expression of a heterologous gene, also known as a transgene, for herbicide tolerance in a plant can confer herbicide tolerance on the plant. However, the expression of a transgene, and therefore its effectiveness, may be influenced by many different factors including the orientation and composition of the cassette driving expression of the individual transgene transferred to the plant chromosome and the chromosomal location and the genomic result of the transgene insertion. This is complicated further in transgenic plants with multiple molecularly-linked transgenes, each conferring a separate trait. In such a situation, proper expression of each of the molecularly-linked transgenes in the plant must result from the same transgene insertion (also called a multi-gene event). In such cases, it is necessary to design and test multiple expression cassettes, each with a different configuration of transgenes and expression elements, and then to produce and analyze a large number of individual plant transformation events through multiple generations of plants in order to select the transgenic event having superior properties relative to the each of the desirable traits and the optimal phenotypic and agricultural characteristics necessary to make it suitable for commercial purposes. Such selection requires extensive molecular characterization as well as greenhouse and field trials over multiple years, in multiple locations, and under a variety of conditions so that a significant amount of agronomic, phenotypic, and molecular data may be collected. The resulting data and observations must then be analyzed by teams of scientists and agronomists with the goal of selecting the event suitable for commercial agricultural use across a wide range of germplasm and in a variety of field conditions. Once selected, the commercial event conferring the desirable traits may be introgressed into other genetic backgrounds using plant breeding methods, thus producing a number of different crop varieties that contain the desirable trait and are suitably adapted to specific local growing conditions.
To make a transgenic plant containing a single transformation event, a portion of a recombinant DNA construct is transferred into the genome of a maize cell using plant transformation techniques. This maize cell is subsequently used to produce a unique R0 plant, which can then be used to produce transgenic progeny plants. The genome of the progeny plants contains the unique event, and these plants can be tested for the desired trait(s) as well as for agronomic performance. The effectiveness of an event can be impacted by cis and/or trans factors relative to the integration site in the transformation event. The phenotype conferred by the event can also be impacted by the size and design of the DNA construct, which can vary by the combination of genetic elements in an expression cassette, number of transgenes, number of expression cassettes, and configuration of such elements and such cassettes. The performance of a given event can be further complicated by factors such as plant developmental, diurnal, temporal, or spatial patterns of transgene expression; or by extrinsic factors, for example, environmental plant growth conditions, water availability, nitrogen availability, heat, or stress. Thus, the ability to create an event conferring a desirable set of phenotypic traits is not readily predictable.